Intermediates for preparing octapeptide vasopressin antagonists

ABSTRACT

Certain octapeptides, which have structures characterized by being a six unit cyclic peptide ring with a dipeptide tail which lacks a glycine unit at position 9, have potent vasopressin antagonist activity. The compounds here claimed are in general characterized by having an amino acid unit at position 4 which is other than valine. An important species of the group is [1-(β-mercapto-β,β-cyclopentamethylenepropionic acid)-2-(O-ethyl-D-tyrosine)-4-α-aminobutyric acid-8-arginine-9-desglycine]vasopressin.

This is a divisional of Ser. No. 624,542, filed June 26, 1984 and nowU.S. Pat. No. 4,542,124, which was, in turn, a continuation-in-partapplication of Ser. No. 467,117, filed Feb. 16, 1983, now U.S. Pat. No.4,469,679 issued Sept. 4, 1984.

This invention relates to cyclic octapeptides which are vasopressinantagonists. The compounds of this invention are 9-desGly-vasopressin(VSP) antagonists or 9-desGly-1-Pmp-vasopressins. More specifically, thestructures of these octapeptides have aβ-mercapto-β,β-cycloalkylenepropionic acid and five amino acid unitscyclized into a 6-unit ring by means of a sulfur derived from a cysteineunit and a sulfur from the propionic acid unit. The ring also has adistinguishing dipeptide tail, which lacks a glycine unit, attached bymeans of an amido linkage to the 6-cysteine unit.

BACKGROUND OF THE INVENTION

M. Manning, W. H. Sawyer and coworkers have published a series ofpublications describing various[1-(β-mercapto-β,β-cyclopentamethylenepropionicacid)-4-valine]-arginine-vasopressin congeners which haveantivasopressin activity. Representative of these are EPA No. 61,356,U.S. Pat. No. 4,367,225 and U.S. Pat. No. 4,399,125.

All of the Manning compounds have a tripeptide chain attached at unit 6and are, of course, nonapeptides. The present compounds aredistinguished over these by being octapeptides, by having a des-Glydipeptide tail attached at unit 6 and by having potent vasopressinantagonist activity.

The potent biological activity of the compounds of the present inventionis unexpected in view of the fact that des-glycinamide⁹ -vasopressin anddes-lysine⁸ -desglycinamide⁹ -vasopressin [T. Barth et al., CollectionCzechoslov. Chem. Commun. 39, 506 (1974)] as well as desglycine⁹-oxytocin [B. Berde et al., Handb. Exp. Pharm 23 860 (1968)] retainlittle of the activity of their respective parent compounds. In fact,Barth reports that desglycinamide⁹ -AVP has CNS activity but practicallyno antidiuretic or uterotonic activity, Belgian Pat. No. 896,509.

Certain of the peptide art designations used in the specification andclaims are the following: Cap, β-mercapto-β,β-cycloalkylenepropionicacid; Pmp, β-mercapto-β,β-cyclopentamethylenepropionic acid; Chg,cyclohexylglycine; Abu, α-amino-n-butyric acid; Cha, cyclohexylalanine;Pba, aminophenylbutyric acid, Gln, glutamine; Gly, glycine; Tyr,tyrosine; Phe, phenylalanine; Phe(4'-Alk), lower alkylphenylalanine;Val, valine; Nva, norvaline; Ile, isoleucine; Nle, norleucine; D-aIle,D-allo-isoleucine; Leu, leucine; Ala, alanine; Lys, lysine; Arg,arginine; Asn, asparagine; Met, methionine; Tos, tosylate; HF, hydrogenfluoride; BHA, benzhydrylamine; DIEA, diisopropylethylamine; 4-MeBzl,4-methylbenzyl; TFA, trifluoroacetic acid; DCC,dicyclohexylcarbodiimide; HBT, 1-hydroxybenzotriazole; ADH, antidiuretichormone; ACM, acetamidomethyl; DMAP, dimethylaminopyridine.

When the term "vasopressin" is used, in the specification only, it meansL-arginine vasopressin (AVP) unless otherwise modified. The AVPderivatives of this invention are preferred. "Alk" represents a loweralkyl of 1-4 carbons which is optionally attached to the nitrogen at Y,to the oxygen substituent of the tyrosine unit when such is present atposition 2 or to the phenyl ring of a phenylalanine unit at ringposition 3. Such alkyl substituents include methyl, ethyl, n-propyl,isopropyl or butyl.

Therefore, in the description herein and in the claims, the nomenclaturecommon in the art of peptide and vasopressin chemistry is used. When noconfiguration is noted, the amino acid unit is in the L, or naturallyoccurring, form.

DESCRIPTION OF THE INVENTION

The desGly⁹ compounds of the invention are illustrated by the followingstructural formula: ##STR1## in which: P is Phe or Phe(4'-Alk);

X is D-Phe, D-Val, D-Nva, D-Leu, D-Ile, D-aIle, D-Pba, D-Nle, D-Cha,D-Abu, D-Met, D-Chg, D or L-Tyr or D or L-Tyr(alk);

Y is NH₂, NHAlk, NHBzl or OH;

W is D-Pro, L-Pro or ΔPro (dehydro-Pro);

A is Val, Ile, Abu, Ala, Gly, Lys, Cha, Nle, Phe, Leu, Chg or Nva;

Z is D-Arg, L-Arg, D-Lys or L-Lys;

n is 0, 1 or 2, or a pharmaceutically acceptable salt, ester prodrug orcomplex thereof.

A subgeneric group of compounds of this invention comprises compounds offormula I in which X is D-Tyr, D-Cha, D-Phe, D-Ile, D-Leu, D-Val orD-Tyr(Et); P is Phe or Phe(4'-Et), A is as defined above, Y is NH₂ ; Wis Pro, Z is Arg and n is 1.

The compounds of formula I in which X is D-Tyr(Et) are particularlyactive ADH antagonists as are the amide⁸ congeners.

Individual compounds of interest are[1-(β-mercapto-β,β-cyclopentamethylenepropionicacid)-2-D-tyrosine-4-valine-8-arginine-9-desglycine]vasopressin,[1-(β-mercapto-β,β-cyclopentamethylenepropionicacid)-2-D-tyrosine-4-valine-8-arginine-9-desglycinamide]vasopressin and,especially, [1-(β-mercapto-β,β-cyclopentamethylenepropionicacid)-2-(O-ethyl-D-tyrosine)-4-valine-8-arginine-9-desglycine]vasopressin.

Also included in this invention are various derivatives of the compoundsof formula I such as addition salts, prodrugs in ester or amide form andcomplexes. The addition salts may be either salts with pharmaceuticallyacceptable cations such as NH₄.sup.⊕, Ca.sup. ○+ + , K.sup.⊕ or Na.sup.⊕at the terminal acid group (Y=OH) or with a pharmaceutically acceptablesalt at a basic center of the peptide (as in the Arg units). The acetatesalt forms are especially useful although hydrochloride, hydrobromideand salts with other strong acids are useful. The compounds, also, forminner salts or zwitter ions as when Y is OH. The ester prodrug formsare, for example, lower alkyl esters of the acids of formula I whichhave from 1-8 carbons in the alkyl radical or aralkyl esters such asvarious benzyl esters. Other latentiated derivatives of the compounds offormula I will be obvious to those skilled in the art "Complexes"include various solvates such as hydrates or alcoholates or those withsupporting resins, such as a Merrified resin.

The compounds of formula I are prepared by cyclizing a linearoctapeptide by means of the two mercapto groups, at the cysteine unit(Cys) at position 6 and at the β-mercapto-β,β-cycloalkylenepropionicacid unit (Cap) at position 1. The cyclization reaction occurs readilyin the presence of a mild oxidizing agent capable of oxidizing amercaptan to a disulfide. The reaction is represented as follows:##STR2## in which: X, P, A and Y are as defined for formula I, above;

Z is as defined for formula I above or also may be a single bondwhenever Y is OH;

W is as defined for formula I above or also may be OH whenever Z and Yare absent; and

Q¹ and Q² are, each, hydrogen or a displaceable group.

The intermediates of formula II are new compounds and are a part of thisinvention. The compounds of formula III in which either or both W and Zare absent are also new compounds useful as intermediates as describedbelow. The latter have VSP antagonist activity at a lower level thanthat of the octapeptides.

The cyclization reaction of this reaction sequence is most usefullycarried out by oxidation. Any oxidizing agent known to the art to becapable of converting a dimercaptan to a disulfide may be used.Exemplary of such agents are an alkali metal ferricyanide, especiallypotassium or sodium ferricyanide, oxygen gas, diiodomethane or iodine.

As an example, potassium ferricyanide is added to the dimercaptan offormula II dissolved in a suitable inert solvent, for example, water oraqueous methanol at temperatures of from 0°-40°. Often, oxidation is ata pH of 7-7.5 at ambient temperature in dilute solution gives goodyields, 40-50%, of the cyclic compound.

The compounds of formula III which are the Cys(OH)⁶ or Pro(OH)⁷compounds are reacted with a dipeptide, a protected (NH₂)-WZY, or anamino acid, (NH₂)-Z-Y, respectively, as described hereafter.

The linear mercaptan starting material may or may not have displaceableor protective groups common to the art (Q¹ and Q²) present at thevarious amino acid units. Such protective groups include benzyl,p-methoxybenzyl, 1-adamantyl, t-butyl, p-nitrobenzyl, trityl,benzylthiomethyl, ethylcarbamoyl or acetamidomethyl. Benzyl, adamantylor t-butyl are removed by mercuric (halo) acetate salts in aqueousmethanol at 0°-80°. The protective group is usually removed beforecyclization such as during the hydrogen fluoride splitting of thepeptide from the supporting resin. It may, however, be removed eitherduring the cyclization or, in situ, before cyclization.

The S-acetamidomethyl groups are especially useful. For example,S-ACM-Pmp-D-Tyr(Et)-Phe-Val-Asn-S-ACM-Cys-Pro-OBzl was treated withpotassium carbonate in aqueous methanol to give the Pro acid linearpeptide in 78-84% yield. This was, then, oxidatively cyclized usingiodine in aqueous methanol to give the desired Pro(OH)⁷ product in65-70% yield. Alternatively, the protected product was cyclized underthe same conditions with initial iodine treatment followed by potassiumcarbonate removal of the protective ester radical. The Pro⁷ acid was,then, condensed with Arg(NH₂), using DCC and DMAP in DMF at 0°-20° togive the ##STR3## in 45% yield.

Iodine, therefore, removes the S-protective group, especially the ACMgroup, and cyclizes the intermediate. Mercuric acetate or lead acetatealso remove the ACM group to yield a metal mercaptide. This is convertedto the thiol in situ by treatment with hydrogen sulfide and, then,oxidized in a separate step.

The desired cyclic octapeptide of formula I can be conveniently isolatedby acidifying the aqueous oxidation mixture, such as using glacialacetic acid, and passing the reaction mixture over an ion-exchangechromatographic column, for example, over a weakly acid, acrylic resincolumn with acid elution, or by gel filtration over a bead-formed gelprepared by cross-linking dextran with epichlorohydrin.

As an alternative to the cyclization of the linear intermediates offormula II suggested above, the cyclized 6-Cys acids or 7-Pro acids(those of formula I in which either both tail units, W and Z, or onlyone tail unit, Z, are absent) are condensed with a protected dipeptide,W-Z-Y, or with an amino acid, Z-Y, respectively. The reaction of the Cysacid or the Pro acid with a suitably protected dipeptide or amino acidis carried out using any amide forming reaction common to the peptideart. Usually, substantially equimolar quantities of the startingmaterials are reacted in the presence of a carbodiimide, such asdicyclohexylcarbodiimide, plus 1-hydroxybenzotriazole ordimethylaminopyridine in an organic solvent at from 0°-35°, preferably,from ice to room temperature. The protective groups are removed by areaction which will not split the disulfide bond of the hexapeptidering, for example, mild alkali.

The important intermediates of formula II are conveniently preparedusing solid-phase methods of peptide synthesis as discussed in M.Manning et al., J. Med. Chem. 25 46 (1982). A commercial benzhydrylaminesupport resin (BHR) is used to prepare the end products of formula I inwhich Y is NH₂ (the des-glycines) and a chloromethyl support resin (CMR)is used to prepare the compounds of formula I in which Y is OH (thedes-glycinamides).

The peptide chain of the linear peptides of formula II is build up,stepwise, proceeding from unit 8 working toward unit 1. Each unit isproperly protected as known in the peptide art and as described below.Alternatively, various oligopeptides may be built up using liquid orsupport reactions, then condensed as a last step in the reactionsequence for preparing the dimercapto intermediates.

The preferred sequence of resin supported step reactions is convenientlycarried out in a Beckman 990B peptide synthesizer without isolation ofeach intermediate peptide. The details of the procedure are in theworking examples presented hereinafter. Solution or enzyme reactionconditions are applicable here as known to the art.

The various amino acids, which are consecutively added to the resinsupported chain are protected as known to the art. For example, the Bocprotecting group is used for an amino group especially at theα-position; an optionally substituted benzyl, for the mercapto groups atthe Pmp and Cys units; tosyl, for the Arg unit; and an optionallysubstituted carbobenzoxy(Z) for the Tyr or Lys units. The protectivegroups should, most conveniently, be those which are easily removed,that is, using acid treatment for the tert.-butyloxycarbonyl group,sodium-liquid ammonia or catalytic hydrogenation for the benzyl orcarbobenzoxy groups where the removal reaction conditions are notconducive to reaction at other portions of the peptide such as thedisulfide bond.

As other examples of protecting groups, the amino group of an amino acidor oligopeptide is protected conventionally by an acyl group such asformyl, trifluoroacetyl, phthaloyl, p-toluenesulfonyl oro-nitrophenylsulfonyl group; a benzyloxycarbonyl group such asbenzyloxycarbonyl, o-bromobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,o- or p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl orp-methoxybenzyloxycarbonyl, an aliphatic oxycarbonyl group such astrichloroethyloxycarbonyl, t-amyloxycarbonyl, t-butoxycarbonyl ordiisopropylmethoxycarbonyl, or an aralkyloxycarbonyl group such as2-phenylisopropoxycarbonyl, 2-tolylisopropoxycarbonyl or2-p-diphenylisopropoxycarbonyl. Amino groups are also protected byforming enamines by reaction with a 1,3-diketone such as benzoylacetoneor acetylacetone.

The carboxyl groups can be protected by amide formation, hydrazideformation or esterification. The amide group is substituted, ifnecessary, with a 3,4-dimethoxybenzyl or bis-(p-methoxyphenyl)-methylgroup. The hydrazide group is substituted with a benzyloxycarbonyl,trichloroethyloxycarbonyl, trifluoroacetyl, t-butoxycarbonyl, trityl or2-p-diphenyl-isopropoxycarbonyl group. The ester group is substitutedwith an alkanol such as methanol, ethanol, t-butanol orcyanomethylalcohol; an aralkanol such as benzylalcohol,p-bromobenzylalcohol, p-chlorobenzylalcohol, p-methoxybenzylalcohol,p-nitrobenzylalcohol, 2,6-dichlorobenzylalcohol, benzhydrylalcohol,benzoylmethylalcohol, p-bromobenzoylmethylalcohol orp-chlorobenzoylmethylalcohol; a phenol such a 2,4,6-trichlorophenol,2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol or2,4-dinitrophenol; or a thiophenol such as thiophenol orp-nitrothiophenol. The hydroxy group in tyrosine is optionally protectedby esterification or etherification. A group protected by esterificationis, for example, an O-acetyl group; a O-benzoyl group,O-benzyloxycarbonyl or O-ethyloxycarbonyl. A group protected byetherification is, for example, an O-benzyl, O-tetrahydropyranyl orO-t-butyl group.

The amino group in the guanidino group in arginine can be protected by asalt forming, nitro, tosyl, benzyloxycarbonyl or mesitylene-2-sulfonylgroup. However, it is not always necessary to protect the guanidinogroup.

The protected linear peptide intermediate is split from the carryingresin matrix, for example, by using ammonia in an alcoholic solvent,and, then, is treated to remove the protective groups, such as by usingsodium-liquid ammonia. This procedure gives the amide derivative of thelinear octapeptide.

More conveniently, the two steps are combined by treating the resinsupported peptide with anhydrous hydrogen fluoride in the presence of asuitable cation scavenger as known to the art, such as anisole, to givethe octapeptide intermediate of formula II, in dimercaptan form, and ingood yield.

The compounds of this invention have potent vasopressin antagonistactivity. Vasopressin is known to contribute to the anti-diureticmechanism of action within the kidney. When the action of thesecompounds antagonizes that of the natural anti-diuretic hormone (ADH),the body excretes water due to an increased permeability of the terminalportions of the renal tubule. We believe the mechanism of action is atthe vasopressin receptors (V₂ -receptors) located on the plasma membraneof certain renal epithelial cells. The most notable pharmocodynamiceffect of the ADH antagonists of the invention is that of a waterdiuretic rather than of a natriuretic such as a thiazide.

Any patient suffering from the syndrome of inappropriate antidiuretichormone secretion (SIADH) or from an undesirable edematous condition isa target for the claimed compounds. Examples of clinical conditionsindicated for the compounds of this invention include hypertension,hepatic cirrhosis, congestive heart failure or a component of anytraumatic condition resulting from serious injury or disease in whichthe agonism of naturally occurring vasopressin at the VSP-mediatedreceptor sites is a contributing factor.

The second group of vasopressin receptor sites are the vascular pressorsites (V₁ -receptors) located within the cardiovascular system itself.For example, Compound 5 of Table I below was tested in the Dyckesprotocol (U.S. Pat. No. 4,367,255) for inhibition of vasopressin-inducedvasoconstriction in the rat; in vitro (pA₂ 8.40) and in vivo (pA₂ 7.71).Antagonism at the V₂ receptor sites results in vasodilation with an endresult of anti-hypertensive activity. Treatment of dysmenorrhea isanother utility for the compounds of this invention when administeredintravenously or intranasally.

The compounds of this invention, therefore, are used to treat edema orto expell water in patients in need of such treatment by administeringparenterally or by insufflation a nontoxic but effective quantity of thechosen compound, preferably combined with a pharmaceutical carrier.Dosage units of the active ingredient are selected from the range 0.01to 10 mg/kg, preferably 0.01 to 5 mg/kg, based on a 70 kg patient. Thedosage units are applied from 1 to 5 times daily.

The pharmaceutical composition for inducing vasopressin antogonismcontains an active ingredient of formula I in the form of a dosage unitas described above dissolved or suspended in a standard liquid carrier.A standard carrier is isotonic saline, contained in an ampoule or amultiple dose vial which is suitable for parenteral injection such asfor intravenous, subcutaneous or intramuscular administration. Acomposition for insufflation is similar but is usually administered in ametered dose applicator or inhaler. Pulverized powder compositions may,also, be used, along with oily preparations, gels, buffers for isotonicpreparations, emulsions or aerosols, as standard composition forms.

The compounds of this invention have been demonstrated to have uniqueantagonistic activity toward the natural antidiuretic hormone (anti-ADHactivity), in vitro, in the medullary tissue of hog or human kidney and,in vivo, in the hydropenic rat or the hydropenic monkey. Details of thein vitro protocols are in F. L. Stassen et al., J. of Pharm. Exp. Ther.233, 50-54 (1982) but the calculations of cyclase activity and bindingpotential at the receptor site are as follows:

Test Procedure for Assay of Adenylate Cyclase Activity:

In each experiment the amount of ³² P/cAMP formed in the absence ofmedullary membrane is determined (blank). The blank value is subtractedfrom all experimental data. The compound is tested for its effect onbasal adenylate cyclase activity and/or on vasopressin stimulatedactivity. Each determination is carried out in triplicate. The Ka valueis derived from a Lineweaver-Burke plot. Rel. V_(max) =(V_(max)drug/V_(max) vasopressin)×100. K_(i) =I/[(Ka'/Ka)-1] where I is theconcentration of the antagonist, and Ka' and Ka are the concentrationsof vasopressin required to give half-maximal activity of adenylatecyclase in the presence and absence of antagonist, respectively.

Test Procedure for Binding Assay:

In each experiment, the amount of ³ H-vasopressin bound in the absenceand in the presence of an excess of vasopressin (7.5×10⁻⁶ M) is measuredin triplicate. These values represent total and non-specific binding,respectively. The K_(B) of a compound is derived from the equation forcompetitive inhibition: K_(B) =IC₅₀ /(1+L/K_(D)), where IC₅₀ is theconcentration required for 50% inhibition of specific ³ H-vasopressinbinding, L is the concentration of the ligand, and K_(D) is thedissociation constant of ³ H-vasopressin (K_(D) =3.6×10⁻⁹ M; 1SD=0.4×10⁻⁹ M). This is the average K_(D) value determined on 3preparations of hog kidney membranes.

Hydropenic Rat Protocol

Food and water are removed from male rats approximately 18 hours priorto testing. Animals are housed 4 per metabolism cage. At 0 hour, thetest compound is administered intraperitoneally to the test group and anequivalent volume of vehicle is administered to both control groups(fasted and non-fasted). Urine volume and osmolality are measured everyhour for 4 hours. Test values are recorded as ml of urine excreted(cumulative), mEq/rat electrolyte excreted, mg/rat urea excreted, andosmolality in milli-Osmoles/kg H₂ O. A tolerance test is used todetermine significance. ED₃₀₀ is defined as the dose of compound (μg/kg)required to lower urine osmolality to 300 m-Osmoles/kg. ED₅₀₀ is definedas the dose of compound (μg/kg) required to lower urine osmolality to500 m-Osmoles/kg. The hydropenic monkey protocol is similar.

                  TABLE I                                                         ______________________________________                                         ##STR4##                                                                                     anti-ADH activity                                                             in vivo                                                                       (Rat)                                                                         ED.sub.300                                                                           in vitro (Pig)                                         X        Y         A      (μg/kg)*                                                                          Ki(nM) K.sub.B (uM)                          ______________________________________                                        1. D-Tyr GlyNH.sub.2                                                                             Val    32     30     0.082                                 2. D-Tyr NH.sub.2  Val    63     27     0.065                                 3. D-Tyr OH        Val    156    160    0.35                                  4. D-Tyr(Et)                                                                           GlyNH.sub.2                                                                             Val    9.9    5.9    0.011                                 5. D-Tyr(Et)                                                                           NH.sub.2  Val    5.8    3.0    0.0078                                6. D-Tyr(Et)                                                                           NH.sub.2  Abu    13     7.6    0.018                                 ______________________________________                                         *Estimated dose of peptide delivered ip stat (μg/kg) which results in      reduction of U.sub.osm from hydropenic levels to 300 mOsmoles/kg H.sub.2      O.                                                                       

Table I demonstrates, in the described protocols, the anti-vasopressinactivity of selected representative compounds whose octapeptidestructures have the desGly dipeptide tail which is characteristic of thecompounds of this invention. Presence of substantial antagonisticactivity is unexpected because, in the agonist series, thedes-Gly-oxytocin has an opposite effect on blood prssure compared withoxytocin itself (See B. Berde et al., loc. cit.) and shortening thelinear tail of oxytocin and vasopressin result is known in the art tocause "a striking decrease of the typical biological activities of thesubstances" (see T. Barth et al., loc. cit.).

Compound 5 of Table I, furthermore, has proven to be a compound ofexceptional antagonist activity across the various testing protocols inhog or human tissue in vitro tests as well as in hydropenic rat andmonkey tests. Its anti-ADH activity, manifested as the dose required todecrease urine osmolality to 300M Osm/kg water in the conscioushydropenic squirrel monkey test, is ED₃₀₀ =8.6 Nmoles/kg (i.e.). That ofCompound 4 of Table I is 33.1 Nmoles/kg. The 2-D-Phe analog of thelatter compound is 319.0 Nmoles/kg.

The following examples are intended solely to teach the preparation ofthe compounds of this invention. All temperaturs are in degreesCentigrade.

EXAMPLE 1 Solid-Phase Synthesis ofPmp(Bzl)-D-Tyr(Br-Z)-Phe-Val-Asn-Cys(OMe-Bzl)-Pro-Arg(Tos) resin

For the solid-phase synthesis of the titled resin supported peptide,Boc-Arg(Tos) resin (3 mmol/5.4 grams of resin) was used as startingmaterial. The appropriately protected amino acids were coupledsequentially onto the Boc-Arg(Tos) resin, prepared by reactingBoc-Arg(Tos) as the cesium salt with commercial Merrifield resin (Cl-CH₂resin) as known to the art, by using a manual program as described inthe following steps:

1. washed with methylene chloride (3 times, 1 minute).

2. prewashed with 33% trifluoroacetic acid in methylene chloride with 1%indole (1 time, 1 minute).

3. deprotection with 33% trifluoroacetic acid in methylene chloride with1% indole (20 minutes).

4. washed with methylene chloride (3 times, 1 minute).

5. prewashed with 10% triethylamine in methylene chloride (1 time, 1minute).

6. neutralization with 10% triethylamine in methylene chloride (10minutes).

7. washed with methylene chloride (3 times, 1 minute).

8. protected amino acid (10 mmol) in triethylamine in methylene chlorideand 0.5M N,N'-dicyclohexylcarbodiimide in methylene chloride (20 ml)were added.

The reaction time was up to two hours.

In the case of the coupling of the Asn moiety, 1-hydroxybenzotriazole(HBT, 10 mmol) was added with Boc-Asn in dry dimethylformamide. Drydimethylformamide (DMF) was also used as solvent when Pmp(Bzl) wascoupled onto the peptide resin, using 4-dimethylaminopyridine (10 mM).Completion of each coupling reaction was monitored by the ninhydrintest. The 4-methoxybenzyl group was used to protect the thiol group ofCys and the 2-bromo-carbobenzoxy group was employed to block thephenolic hydroxyl of D-Tyr.

The resulting protectedPmp(Bzl)-D-Tyr(Br-Z)-Phe-Val-Asn-Cys(OMe-Bzl)-Pro-Arg(Tos)-resin waswashed well with methylene chloride and methanol, respectively. Afterdrying in vacuo overnight, 8.4 grams of the titled protected resinintermediate was collected. ##STR5##

Pmp(Bzl)-D-Tyr-(p-bromocarbobenzoxy)-Phe-Val-Asn-Cys(OMe-Bzl)-Pro-Arg(Tos)resin (4 g, ca. 1.5 mmol) was subjected to ammonolysis using saturatedammonia/methanol solution (200 ml) in dry dimethylformamide (50 ml) atroom temperature for 48 hours. After evaporation to dryness, the residuewas precipitated by ethyl acetate/n-hexane and filtered to give theprotected octapeptide amide (1.54 g).

This crude peptide was dissolved in liquid ammonia (250 ml) and treatedwith sodium/liquid ammonia solution to givePmp-D-Tyr-Phe-Val-Asn-Cys-Pro-Arg-NH₂ which was, then, oxidized using0.01M potassium ferricyanide solution in 4 l. of aqueous solution at pH7-7.5. After the completion of oxidation reaction, the pH of aqueoussolution was adjusted to pH 4.5 by adding glacial acetic acid. Thissolution was passed through a weakly acid acrylic resin (Bio-Rex 70)column (11×2.5 cm, H⁺ form) slowly. The column was eluted with 5% and50% acetic acid solution, respectively. Crude cyclized ##STR6## wascollected from 50% acetic acid solution fractions (860 mg).

    ______________________________________                                        Purification of                                                                ##STR7##                                                                     ______________________________________                                        1.  Counter-current distribution:                                             Sample:  860 mg crude, n-BuOH/HOAc/H.sub.2 O (4:1:5) 250                               transfers                                                            (a)      fr. 186-204,    436 mg                                               (b)      fr. 182-185 & 205-218,                                                                        219 mg                                               2.  Partition chromatography:                                                 Sample:  250 mg (from 1-a),                                                                          G-25 fine (2.5 × 55 cm),                                                n-BuOH/HOAc/H.sub.2 O (4:1:5)                          (a)      fr. 32-46       222 mg                                               3.  Preparative HPLC:                                                         Sample:  40 mg (from 2-a); Alltech C18, 3000 psig. Flow                                rate: 3.0 ml/min.                                                                 Buffer A: 0.1% TFA                                                            Buffer B: 0.25% TFA/CH.sub.3 CN (4:6)                                         60% B; isocratic; 235 nm (2.0 AUFS)                                           Injection: 10 mg/0.5 ml. buffer A                                       17 mg of pure titled compound.                                         4.  Ion-exchange Chromatography:                                              Sample:  365 mg (from 1-a & 2-a); CMC; 0.01M NH.sub.4 OAc                              to 0.1M NH.sub.4 OAc                                                                          Linear gradient                                      (a)      fr. 51-70       93.5 mg                                              (b)      fr. 71-89       86.5 mg                                              (c)      fr. 91-110      65mg                                                 (d)      fr. 111-121     24.5 mg                                              ______________________________________                                    

EXAMPLE 2 ##STR8##

Pmp(Bzl)-D-Tyr(Br-Z)-Phe-Val-Asn-Cys(OMe-Bzl)-Pro-Arg(Tos)-Resin (4.2 g,1.5 mmol) from Example 1, in 4.5 ml distilled anisole, was reacted withanhydrous hydrogen fluoride (40 ml) at 0° for one hour. After treatmentas described above and evaporation in vacuo to dryness, the residue wastreated with anhydrous ether and filtered off to give 1.33 g crudepeptide. The completion of removal of the Bzl group from the Pmp moietywas carried out using the sodium in liquid ammonia reaction as describedin Example 1. The resulting unprotected octapeptide was cyclized using0.01M potassium ferricyanide solution at pH 7-7.5 until color persistedfor 30 minutes again as described above in the preparation of the amide.

Desglycinamide octapeptide (600 mg) was collected after acidifying theoxidation solution with acetic acid to pH 4.5 and passing the reactionmixture over a Bio-Rex-70 column with 1 l. of 5% acetic acid as eluent.

    ______________________________________                                         ##STR9##                                                                 

    ______________________________________                                        1.  Counter-current distribution:                                             Sample:  600 mg from Bio-rex 70. n-BuOH/HOAc/H.sub.2 O                                 (4:1:5); 200 transfers                                               (a)      fr. 150-161     169 mg                                               (b)      fr. 133-149 & 162-163                                                2.  Preparative HPLC:                                                         Sample:  52 mg (from 1-a); Alltech C18 (25 cms 10 mm,                                  10 micron);                                                                       Buffer A: 0.1% TFA                                                            Buffer B: 0.25% TFA/CH.sub.3 CN (4:6)                                         60% B, isocratic; 3000 psig;                                                  3.0 ml/min.                                                                   Injection: 10 mg/0.6 ml in buffer A                                           235 nm (2.0 AUFS).                                               (a)      24 mg                                                                (b)      7.3 mg                                                               Combine 2-a and 2-b, repurified on HPLC to give 15 mg                         pure peptide.                                                                 3.  Partition Chromatography:                                                 Sample:  117 mg (from 1-A),                                                                          G-25 fine (2.5 × 55 cm)                                                 n-BuOH/HOAc/H.sub.2 O 4:1;5                            (a)      fr. 32-36       83 mg of pure product                                ______________________________________                                    

EXAMPLE 3 ##STR10##

The titled compound was prepared by the solid phase method onbenzhydrylamine resin (BHA). Thus, 1.0 g BHA resin (1.13 mmol NH₂ /gresin) was reacted with 1.5 equivalents of Boc-Arg(Tos), 1.5 equivalentsof DCC and 3.0 equivalents of HBT which were made up indimethylformamide to be 0.1M in Boc-Arg(Tos). Deblocking was performedwith 50% TFA/methylene chloride and neutralization with 5%DIEA/methylene chloride. The peptide was elongated, stepwise, bycoupling, using preformed Boc aminoacyl symmetrical anhydrides in DMF(0.1M). Boc-Asn, Boc-D-Tyr(Et) and Pmp(MBz) were successively coupledusing DCC and HBT in DMF. Completeness of coupling was monitored by thequalitative ninhydrin test and recoupling was performed as necessary.The completed Pmp(MBz)-D-Tyr(Et)-Phe-Val-Asn-Cys(MBz)-Pro-Arg(Tos)-BHAresin was washed with methylene chloride and dried to constant weight,2.34 g.

The peptide was deblocked and cleaved from the resin by treatment withanhydrous liquid hydrogen fluoride (30 ml) in the presence of anisole (4ml) at 0° for one hour. After evaporation to dryness under vacuum, theresin was washed with ethyl ether, air dried and, then, extracted withdegassed dimethylformamide (3×20 ml) and 20% acetic acid (4×20 ml). TheDMF and acid extracts were added to 4 l of water (pH 4.5 with aceticacid). The pH was adjusted to 7.2 with ammoniium hydroxide and thesolution was titrated with 0.01M potassium ferricyanide under argon withstirring until a yellow color persisted (85 ml). The pH was brought to4.8 with glacial acetic acid. The mixture was filtered and the filtratepassed over a Bio-Rex 70 column (H.sup.⊕). After washing the column withwater (200 ml) the crude peptide was eluted with 300 ml ofpyridine/acetic acid/water (30:4:66 v/v). The eluant was evaporatedunder vacuum at 30°. The residue was dissolved in 100 ml of 0.2N aceticacid, then, lyophilized, yielding 507 mg of the crude titledoctapeptide.

    ______________________________________                                        Purification of                                                                ##STR11##                                                                    ______________________________________                                        1.  Counter-current distribution:                                             Sample:  607 mg crude, n-BuOH:HOAc:H.sub.2 O, 4:1:5, 240                               transfers                                                            (a)      fr. 154-170 & 190-192                                                                          71 mg                                               (b)      fr. 171-189      230 mg                                              2.  Gel filtration                                                            Sample:  123 mg of Sample (b), G-15 (2.5 × 55 cm) using                          0.2 N HOAc,                                                                   25 ml/hr                                                             (a)      fr. 46-50       ˜20 mg                                         (b)      fr. 51-77        60 mg pure peptide                                  ______________________________________                                    

EXAMPLE 4 ##STR12##

A mixture of 0.1 mmole of (Pmp¹ -D-Leu² -Val⁴ -desGlyNH₂)AVP, preparedas described above but using Boc-D-Leu at position 2, and 0.1 mmole ofn-propylamine in 20 ml of DMF was reacted with 23 mg (0.11 mmol) of DCCand 14 mg (0.11 mmol) of HBT at room temperature for 2 hours. Thevolatiles were evaporated to give an oily product residue. The productwas purified as described above using: (1) gel filtration overG-10-Sephadex eluted with 0.2N acetic acid; (2) high pressure liquidchromatography using 0.05% TFA in 39% acetonitrile in water; and, again,(3) gel filtration to give 20 mg of the pure octapeptide of the title.

Amino acid analysis: Asp 0.88, Pro 0.93, Val 1.00, Leu 1.09, Phe 0.88,Arg 1.07. HPLC=95% major peak at 11.33 with 40% aqueous acetonitrilewith 0.05M KH₂ PO₄ as buffer. K_(bind) =12.1% inhibition at 10⁻⁵ M.

Using (Pmp¹ -D-Tyr(Et)² -Val⁴ -desGlyNH₂)-AVP prepared as in Example 2above and benzylamine gives ##STR13## Other N-alkylated derivatives areprepared similarly.

EXAMPLE 5 Solid Phase Peptide Synthesis ofPmp(4-MeBzl)-D-Tyr(Et)-Phe-Abu-Asn-Cys(4-MeBzl)-Pro-Arg(Tos)-BHA resin

For the solid phase synthesis of the titled resin-supported peptide,Boc-Arg(Tos)BHA resin (1.19 mmol/g of resin) was used as a startingmaterial. It was prepared by reaching Boc-Arg(Tos), 3 mmol, with thebenzhydrylamine resin, 1.0 mmol, in dimethylformamide for two hours. Thebenzhydrylamine resin as the hydrochloride salt was covered withmethylene chloride overnight. It was, then, washed with methylenechloride (4×1 min), neutralized with 7% diisopropylethylamine inmethylene chloride (2×2 min), then, 6×1 min with methylene chloridealone and, finally, 2×1 min with predried dimethylformamide. The loadingof Boc-Arg(Tos) on the resin was carried out twice on the shaker using1-hydroxybenzotriazole (HBT, 6 mmol) and dicyclohexylcarbodiimide (DCC,3 mmol). A quantitative ninhydrin test and amino acid analysis wereperformed routinely after loading to determine the percentage loading onthe resin. Loading in this particular run was 62.66%, i.e. 0.74 mmol/gof resin was available. The subsequent amino acid, Boc-Pro, was coupledon the shaker using the following protocol.

(1) Washed with methylene chloride (6 times, 1 min).

(2) Prewashed with 50% TFA in methylene chloride (1 time, 1 min).

(3) Deprotected with 50% TFA in methylene chloride (20 min).

(4) Washed with methylene chloride (6 times, 1 min).

(5) Prewashed with 7% DIEA in methylene chloride (1 time, 1 min).

(6) Neutralized with 7% DIEA in methylene chloride (8 min).

(7) Washed with methylene chloride (6 times, 1 min).

(8) Washed with dimethylformamide (2 times, 1 min).

(9) Added protected amino acid (3 mmol) and HBT, 6 mmol, in DMF,followed by the addition of DCC in methylene chloride, 3 mmol, andcoupling for 2 hours.

(10) Washed with dimethylformamide (2 times, 1 min).

(11) Washed with methylene chloride (4 times, 1 min).

(12) Washed with ethanol/methylene chloride 1:1 (2 times, 1 min).

(13) Washed with methylene chloride (4 times, 1 min).

The subsequent amino acids were coupled sequentially using Beckmanpeptide synthesizer 990-B. The program used for each coupling exceptBocAsn and Pmp(4-MeBzl) was as follows.

(1) Washed with methylene chloride (3 times, 1 min).

(2) Prewashed with 50% TFA in methylene chloride (1 time, 1 min).

(3) Deprotection with 50% TFA in methylene chloride (30 min).

(4) Washed with methylene chloride (3 times, 1 min).

(5) Prewashed with 7% DIEA in methylene chloride (1 time, 1 min).

(6) Neutralized with 7% DIEA in methylene chloride (1 time, 10 min).

(7) Washed with methylene chloride (3 times, 1 min).

(8) Protected amino acids (3 mmol) in methylene chloride, followed byaddition of DCC, 3 mmol, 10 ml of 0.3M in methylene chloride, andcoupling for two hours.

(9) Washed with methylene chloride (3 times, 1 min).

(10) Washed with ethanol/methylene chloride, 1:1, (3 times, 1 min).

(11) Washed with methylene chloride (3 times, 1 min).

In case of coupling of Asn moiety, 1-hydroxybenzotriazole (HBT, 6 mmol)was used, 10 ml of 0.6M dimethylformamide. Dry dimethylformamide wasalso used as solvent when Pmp(4-MeBzl) was coupled onto the peptideresin, using 4-dimethylaminopyridine (3 mmol). Completion of eachcoupling reaction was monitored by the ninhydrin test. The4-methylbenzyl (4-MeBzl) group was used to protect the thiol groups ofthe Cys and pentamethylene mercaptopropionic acid (Pmp) moieties.

Preparation of Pmp-D-Tyr(Et)-Phe-Abu-Asn-Cys-Pro-ArgNH₂

Pmp(4-MeBzl)-D-Tyr(Et)-Phe-Abu-Asn-Cys-(4-MeBzl)-Pro-Arg(Tos)BHA-resin,1.25 g, (0.37 mmol) in 2 ml of anisole, was reacted with anhydroushydrogen fluoride (20 ml at 0° for 50 min). After evaporation of HF invacuo, the residue was washed with anhydrous ether, 4×20 ml, and thecrude peptide was extracted with dimethylformamide (50 ml) and 33%acetic acid (50 ml) into 2 liter of degassed water previously adjustedto pH 4.5. The aqueous diluted disulfhydryl octapeptide was cyclizedusing 0.01M potassium ferricyanide solution at pH 7.2 until the yellowcolor persisted for 30 minutes (50 ml). The pH was adjusted to 4.5 usingglacial acetic acid and the solution was passed through a weakly acidacrylic resin (Bio-Rex-70) column (2.5×12, H.sup.⊕ form), slowly. Thecolumn was eluted with pyridine-acetate buffer (30:4:66;pyridine/glacial acetic acid/water). The pyridine acetate solution wasremoved by distillation in vacuo. The residue was lyophilized from 10%acetic acid to give 300 mg (76%) of crude titled peptide.

    ______________________________________                                         ##STR14##                                                                

    ______________________________________                                        1.  Counter-current distribution:                                                 Sample: 300 mg, n-BuOH/HOAc/H.sub.2 O, 4:1:5, 240 transfers.                  (a) fr. 176-186, 99.6 mg of pure peptide                                      (b) fr. 170-175 and 187-210, 117.24 mg                                        Yield of purified material, 216.84 mg (55%)                               2.  Molecular Formula:                                                                           C.sub.50 H.sub.72 N.sub.12 O.sub.10 S.sub.2                    Molecular Weight:                                                                            1064.53                                                        Amino Acid Analysis:                                                                         Asp (1.00), Abu + Cys (1.70),                                                 Tyr (0.64), Phe (0.98), Arg                                                   (0.91)                                                         Peptide Content:                                                                             68.06-91.52% from amino acid                                                  analysis 87.33% from nitrogen                                                 analysis                                                   3.  Chromatography                                                                Data:          Solvent            R.sub.f                                     TLC            n-BuOH/HOAc/H.sub.2 O/EtOAc                                                                      0.56                                                       (1:1:1:1)                                                                     n-BuOH/HOAc/H.sub.2 O/                                                                           0.42                                                       (4:1:5) Upper                                                  HPLC           C.sub.18 -column   k'                                          Isocratic      H.sub.2 O/CH.sub.3 CN/TFA,                                                                       3                                                          (60:40:0.25)                                                                  0.05 MKH.sub.2 PO.sub.4 :                                                                        7.33                                                       acetonitrile (60:40)                                           Gradient       H.sub.2 O/CH.sub.3 CN/TFA,                                                                       8.82                                                       80:20:0.25 to                                                                 50:50:0.25                                                     Fast Atom Bombard-                                                                           m/z 1065 (M + H).sup.+ ;                                       ment (FAB):    1063 (M - H).sup.-                                         ______________________________________                                    

EXAMPLE 6 Solid Phase Peptide Synthesis ofPmp-(4-MeBzl)-D-Tyr(Et)-Phe-Ala-Asn-Cys-4-MeBzl)-Pro-Arg(Tos)-BHA resin

The tetrapeptide supported resin, Boc-Asn-Cys(4-MeBzl)-Pro-Arg(Tos)-BHA,0.72 g (0.36 mmol), was synthesized on Beckman 990-B peptidesynthesizer, starting from the Boc-Arg(Tos) benzhydrylamine resin (0.72mmol/g) using a protocol like that of Example 5. The subsequent aminoacids were coupled sequentially on the shaker using HBT and DCC for 2hours in a similar fashion. After coupling of the last residue, i.e,Pmp(4-MeBzl), the resin containing peptide was washed as usual, dried togive 0.88 g of the titled intermediate. ##STR15##

Pmp(4-MeBzl)-D-Tyr(Et)-Phe-Ala-Asn-Cys(4-MeBzl)-Pro-Arg(Tos)-BHA-resin,in 2 ml of anisole, was reacted with anhydrous HF, 20 ml, at 0° for 50minutes. The work up was done as usual and the uptake of K₃ Fe(CN)₆ was45 ml to give 230 mg (60.8%) of crude titled peptide.

    ______________________________________                                         ##STR16##                                                                

    ______________________________________                                        1.  Counter-current distribution:                                                 Sample: 230 mg, n-BuOH/HOAc/H.sub.2 O, 4:1:5, 240 transfers                   (a) fr. 160-178, 105.2 mg pure product                                        (b) fr. 179-190 and 150-159, 49.5 mg                                          Yield of purified material, 154.7 mg (41%).                               2.  Molecular Formula:                                                                           C.sub.49 H.sub.70 N.sub.70 O.sub.10 S.sub.2                    Molecular Weight:                                                                            1050.449                                                       Amino Acid Analysis:                                                                         Asp (1.00), Pro (1.03), Ala                                                   (0.94), Cys (0.46), Tyr (0.65),                                               Phe (0.91), Arg (0.92).                                        Peptide Content:                                                                             59.18-81.77% from two                                                         analyses.                                                  3.  Chromatography                                                                Data:          Solvent            R.sub.f                                     TLC            mBuOH/HOAc/H.sub.2 O/EtOAc                                                                       0.64                                                       (1:1:1:1)                                                      HPLC           C.sub.18 -column   k'                                          Isocratic      H.sub.2 O/CH.sub.3 CN/TFA,                                                                       2.18                                                       60:40:0.1                                                      Gradient       H.sub.2 O/CH.sub.3 CN/TFA,                                                                       6.47                                                       60:40:0.1 to 50:50:0.1                                         Fast Atom Bombard-                                                                           m/z 1051 (M + H).sup.+ ;                                       ment (FAB):    1049 (M - H).sup.-                                         ______________________________________                                    

EXAMPLE 7 Solid Phase Peptide Synthesis ofPmp(4-MeBzl)-D-Tyr(Et)-Phe(4'-Et)-Val-Asn-Cys-(4-MeBzl)-Pro-Arg(Tos)-BHA-resin

The titled resin-supported peptide was prepared from BOC-Arg(Tos) BHAresin (0.4 mmol/g) on a shaker using a protocol used before i.e.deprotection-coupling using HBT and DCC for 2 hours, up toBoc-Val-Asn-Cys-(4-MeBzl)-Pro-Arg(Tos)-BHA resin. The next two aminoacid residues were coupled using the Beckman peptide synthesizer 990-B.The Pmp(4-MeBzl) was coupled manually using DMAP-DCC overnight. Theresin-containing peptide was washed and dried as usual to give 2.00 g ofthe titled intermediate. ##STR17##

Pmp-(4-MeBzl)-D-Tyr(Et)-Phe(4-Et)-Val-Asn-Cys-4-MeBzl)-Pro-Arg(Tos)-BHAresin, in 3 ml of anisole was reacte with 30 ml of anhydrous hydrogenfluoride at 0° for an hour. The work up was done as described above,with 38 ml of K₃ Fe(CN)₆ taken up. About 50 mg of crude peptide wasobtained from the Bio-Rex column and 139 mg was precipitated out ofsolution, total yield 189 mg (42.7%) of titled peptide.

    ______________________________________                                        Purification:                                                                 1.  Partition column chromatography, Sephadex, G-25:                          Sample: 50 mg, n-BuOH/HOAc/H.sub.2 O, 4:1:5,                                  (a)     fr. A, 23.86 mg                                                       (b)     fr. B, 18.5 mg                                                        Preparative HPLC                                                              Sample:  43 mg (From 1, Fr. a + Fr. b), Altex ODS,                                     10 mm × 25 cm, 5μ, flow rate 4 ml/min.,                              water/acetonitrile/TFA (50:50:0.25),                                          isocratic, 229 nm (2.0 AUFS), injection 2.0                                   mg/300 μl and 4.0 mg/420 ml to give 30.0 mg                                of pure peptide.                                                     2.  Physical Data:                                                            Molecular Formula:                                                                           C.sub.53 H.sub.78 N.sub.12 O.sub.10 S.sub.2                    Molecular Weight:                                                                            1106.47                                                        Amino Acid Analysis:                                                                         Asp (1.00), Pro (0.78-0.84), Cys                                              (0.45), Val (1.02), Tyr (0.63),                                               Phe (p-Et) (1.50), Arg (1.00-0.96)                             Peptide Content:                                                                             73.3-89.6%                                                     3.  Chromatography                                                                Data:                                                                         TLC           Solvent             R.sub.f                                                   nBuOH/HOAc/H.sub.2 O/EtOAc,                                                                       0.70                                                      1:1:1:1                                                                       nBuOH/HOAc/H.sub.2 O,                                                                             0.299                                                     4:1:5 Upper                                                     HPLC          C.sub.18 Column     k'                                          Isocratic     H.sub.2 O/CH.sub.3 CH/TFA,                                                                        4.43                                                      55:45:0.1                                                       Gradient      H.sub.2 O/CH.sub.3 CN/TFA,                                                                        8.7                                                       60:40:0.1 to 50:50:0.1                                          FAB           m/z 1107 (M + H).sup.+ ;                                                      1105 (M + H).sup.-                                          ______________________________________                                    

EXAMPLE 8 Synthesis of Boc-Asn-Cys(4-MeBzl)-Pro-Arg(Tos)MBHA resin

One millimole of Boc-Asn-Cys(4-MeBzl)-Pro-Arg(Tos)-BHA resin wasprepared using 1 mmole of Boc-Arg(Tos)-4-methylbenzhydrylamine (MBHA)resin as starting material by coupling sequentially with the appropriatet-Boc-protected amino acids in a Beckman 990-B peptide synthesizer,990-B.

1.83 Grams of the protected peptide resin was obtained and was dividedinto two equal parts of 0.915 g each. ##STR18##

One part of the protected peptide resin from above was furthersequentially coupled with 1.5 mmoles of the appropriate Boc amino acidsand β-(S-MeBzl)-Pmp-OH to give 1.16 g of the final protected peptideresin. Pmp(S-MeBzl)-D-Tyr(Et)-Phe-Gly-Asn-Cys(4MeBzl)-Pro-Arg(Tos)MBHAresin was obtained and dried in vacuo. This protected resin was treatedwith 1.5 ml of anisole and 25 ml of anhydrous hydrogen fluoride at 0°for 1 hour. The deprotected peptide was treated with 0.01 mole ofpotassium ferricyanide solution at pH 7.2 in 2 liters of water. 53 Ml ofthe oxidizing agent was used.

The resulting solution was passed through a C₁₈ flash column. The columnwas eluted with 50% of acetonitrile with 0.25% trifluoroacetic acid in20 ml per fraction. 325 Mg crude product was isolated from thefractions. Further purification of the product by CCD (B/A/W, 4:1:5) toobtain 188 mg of 99% pure titled product.

    ______________________________________                                        Amino acid analysis:                                                          ______________________________________                                        Peptide content       82%                                                     Asp 1.04              Tyr 0.92                                                Pro 1.15              Phe 1.01                                                Gly 1.00              Arg 0.91                                                Cys 0.54                                                                      ______________________________________                                         FAB/MS = m/z (M + H).sup.+ 1037                                          

EXAMPLE 9 ##STR19##

One part of the protected peptide resin from Example 8 was furthersequentially coupled with 1.5 mmoles of the appropriate Boc amino acidsand β-(S-4-MeBzl)-Pmp-OH to give 1.06 g of the final protected peptideresin,Pmp(S-4-MeBzl)-D-Tyr(Et)-Phe-Chg-Asn-Cys(S-4-MeBzl)-Pro-Arg(Tos)MBHAresin, obtained after drying in vacuo.

This protected peptide resin was treated with 1.5 ml of anisole and 25ml of anhydrous hydrogen fluoride.

Following the usual oxidation by potassium ferricyanide and isolationover a C₁₈ column, 165 mg crude titled product was obtained. Furtherpurification by CCD G-15 and P-2 gel filtration as described above gave55 mg HPLC pure titled product.

Peptide content: 88%

FAB/MS: m/z 1119 (M+H)⁺

EXAMPLE 10 ##STR20##

4.87 g (15 mmol) of the BocCys(4MeBzl) was dissolved in 30 ml of ethanoland 10 ml of water added. The pH was then adjusted to 7.1 with anaqueous solution of cesium bicarbonate.

The mixture was concentrated and the residue evaporated three times from50 ml of toluene. This residue was, then, placed under high vacuum atambient temperature overnight.

The salt was dissolved in 35 ml of dimethylformamide and 5 g ofcommercial chloromethylphenyl resin added. The mixture was stirred at53° under argon overnight.

The mixture was filtered and the resin washed with dimethylformamide(5×60 ml), DMF/Water, 9:1, (5×60 ml), DMF (5×60 ml) and ethanol (6×60ml). It was, then, dried under high vacuum at ambient temperature overthe weekend.

The peptide chain was built up in a Beckman synthesizer as describedabove using the Boc derivatives of Asn, Val, Phe, D-Tyr(Et) and theS-(4-MeBzl) Pmp derivative. The resin was removed and placed in a manualshaker.

0.86 G of the peptide resin was treated with 1.5 ml of anisole andstirred for 60 min at 0° in 15 ml of hydrogen fluoride. The hydrogenfluoride was, then, removed under aspirator pressure at 0°.

The residue was then washed with 3×25 ml of ether (discarded) and thepeptide eluted with dimethylformamide and 30% acetic acid (4×10 ml).This solution was added to 21 of degassed water and the pH adjusted to7.0 with ammonium hydroxide. A 0.01M potassium ferricyanide solution wasadded slowly (35 ml).

The pH was then adjusted to 4.5 with acetic acid and the mixture stirredfor 30 minutes with 25 g (WET) of a weakly basic ion exchange resin(AG-3×4 1R-4S). The suspension was filtered and the resin washed with2×400 ml of 30% acetic acid.

The filtrate was, then, passed thru a C₁₈ flash column (7×16 mm). Thecolumn was then washed with water (3×400 ml) and the peptide eluted withacetonitrile/water/TFA, 50:50:0.25). Fractions 30→36 were combined,concentrated and lyophilized to yield 25 mg of the titled freeCys(OH)cyclic intermediate.

FAB mass spectrum in glycerol: 827 (M+H)⁺, 825 (M-H)⁻.

The Cys acid (20 mg) is reacted with one equivalent of Pro-Arg(NH₂)HCl(prepared from the commercial dihydrochloride by treatment with 1equivalent of triethylamine) in the presence of DCC and HBT indimethylformamide to produce the compound of Example 3. Similarly,Pro(OMe) is attached to the Cys acid, hydrolyzed with mild sodiumhydroxide to give the Pro acid which is, then, reacted withArg(HCl)(OMe) to give the acid parent of the compound of Example 3 aftermild hydrolysis of the ester. This compound is isolated as the potassiumsalt if desired. See Example 12 below. Alternatively, the Pro-Arg(NH₂)is used in the condensation directly.

A mixture of 4.5 mg of Pmp-D-Tyr(Et)-Phe-Val-Asn-Cys-OH prepared asabove and 1 ml of methanol was treated with ethereal diazomethane andpurified by preparing HPLC (50% CH₃ CN/50% H₂ O/0.1% TFA) to yield 4.3mg of the methyl ester (94%), FABMS m/z 841 (M+H)⁺, homogeneous by HPLCand TLC.

EXAMPLE 11 ##STR21##

BocPro-Merrifield resin was made by coupling BocPro to Merrifield resinusing the cesium salt method to give Boc-Pro-OCH₂ -C₆ H₄ -resin whichwas used as the starting material for the synthesis. The synthesis wascarried out on the Beckman 990-B peptide synthesizer using the followingprotocol. Three equivalents of the amino acids were dissolved in theirappropriate solvents [the Boc derivatives of 4MeBzl-Cys, Val, Phe inmethylene chloride, Asn in dimethylformamide, X such as D-Tyr(Et) orBrBz-D-Tyr in 1:1 methylene chloride/dimethylformamide and 4MeBzl-Pmp inmethylene chloride] and were coupled using an equimolar amount ofdicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT) exceptfor the coupling of 4MeBzl Pmp where 1.0 equivalent ofdimethylaminopyridine was used as catalyst. The extent of coupling wasdetermined by qualitative ninhydrin analyses and couplings were repeatedwhen necessary. The Boc groups were removed using 1:1 trifluoroaceticacid/methylene chloride and after washing the free amine was generatedusing 5% diisopropylethylamine/methylene chloride. The sequence of thepeptide was checked using solid phase sequencing befor the coupling ofthe 4MeBzl-Pmp and its homogeneity confirmed. After the final coupling,the resin was dried to give 2.24 g of peptide resin in the case of theD-Tyr(Et)² -Pro⁷ compound.

1.1 G (0.5 mmole) of the D-Tyr(Et)² peptide resin with 3 ml of anisolewas stirred 60 min. at 0° (ice bath) in 25 ml of hydrogen fluoride (HF).The HF was, then, removed under reduced pressure at 0°. The residue waswashed with ethyl ether (4×20 ml, discarded) and the peptide eluted withdimethylformamide 3×10 ml, 20% acetic acid 3×10 ml and 0.3N ammoniumhydroxide 3×10 ml.

The filtrate was added to 2 l of degassed water and the pH adjusted to7.1 with conc. ammonium hydroxide. A 0.01M solution of potassiumferricyanide was then added dropwise with stirring until a faint yellowcolor persisted (41 ml). This solution was adjusted to pH=4.7 withacetic acid and stored in the cold overnight.

The solution was adjusted to pH=7 with ammonia and stirred for 15 minwith 30 g of AG-3×4 Bio-Rad ion exchange resin (wet, Cl form). Thissolution was then filtered slowly through an additional 30 g of resin.The resin was then washed with 4×200 ml of 20% acetic acid and thefiltrate stored in the cold overnight.

The filtrate was then passed through a flash column (5 cm×10 cm) of apacking of silica gel coated with a C-18 silane. The column was then,washed with 350 ml of water and the peptide eluted with 500 ml of 1:1acetonitrile/water (0.25% trifluoroacetic acid) in 20 ml fractions.

Fractions 11-17 were combined and concentrated. The residue wasdissolved in conc. acetic acid, diluted with water and lyophilized toyield 189 mg of the D-Tyr(Et)², proline peptide, which was used withoutfurther purification for the synthesis of the tail modified peptides.

    ______________________________________                                        Identification of:                                                            ______________________________________                                         ##STR22##                                                                    Amino Acid Analysis:                                                                       Peptide Content 55%                                                           Asp, 1.00; Pro, 1.23; Cys, 0.35; Val; 1.04,                                   Tyr(Et), 1.43; Phe, 1.51.                                        HPLC:        Satisfactory.                                                     ##STR23##                                                                    Amino Acid Analysis:                                                                       Peptide Content 82%                                                           Asp, 0.97; Pro, 1.10; Cys, 0.39; Val, 1.05;                                   Tyr, 0.99; Phe, 0.99                                             HPLC:        Satisfactory, 30% CH.sub.3 CN/70% 0.05 m                                      KH.sub.2 PO.sub.4, 2 ml/min, 5 uC-18, k' = 6.14.                 ______________________________________                                    

A mixture of 10 mg of the D-Tyr(Et)-Pro(OH)⁷ prepared as above, and 1 mlof methanol was treated with ethereal diazomethane and, then, purifiedby preparing HPLC (50% CH₃ CN/50% H₂ O/0.1% TFA) to yield 7.5 mg of themethyl ester (74%), FABMS m/z 938 (M+H⁺), homogeneous by HPLC and TLC.

To a solution of the D-Tyr(Et)² -proline heptapeptide, prepared asdescribed above, (29.7 mg, 0.0331 mmol), and Arg(NH₂) (0.0996 mmol) indimethylformamide (400 μl), dicyclohexylcarbodiimide (10.3 mg, 0.05mmol) and dimethylaminopyridine (0.05 mmol) were added and the reactionmixture was stirred at 0°-20° for 4 hours. The dimethylformamide was,then, removed under vacuum. The residue was treated as above in Example3 in 45% yield to give the desired D-Tyr(Et)² -Val⁴ amide.

EXAMPLE 12 ##STR24##

The linear peptidyl resin,Pmp(S-MeBzl)-D-Tyr(Et)-Phe-Val-Asn-Cys(S-MeBzl)-Pro-D-Arg(Tos)-BHAresin, was prepared by the solid phase method using the standardprotocol described above. Thus, 1.5 g benzhydrylamine resincorresponding to 1.0 mmol amine was coupled successively with the Bocamino acid derivatives in threefold excess using DCC/HOBt in methylenechloride/DMF, 1:1. Pmp(S-MeBzl) was coupled with DCC/DMAP. Completenessof coupling was checked with the Kaiser test or a quantitative ninhydrintest. Recoupling was performed until the test was negative.

The protected peptidyl resin was washed with successive portions ofmethylene chloride, methanol, ethyl acetate and methylene chloride, and,then, air dried. The peptide was cleaved from the resin with 15 ml ofliquid hydrogen fluoride in the presence of 1.0 ml of anisole at 0° forone hour. After evaporation of the hydrogen fluoride and drying underhigh vacuum, the resin was washed with 3×20 ml of ether and, then,extracted with 2×50 ml of 50% acetic acid, 50 ml of 10% acetic acid, and50 ml of water. The combined extracts were diluted to 4 l with water andthe pH adjusted to 7.2 with 50% sodium hydroxide solution. The solutionwas titrated with 0.01M K₃ Fe(CN)₆ solution until a yellow colorpersisted (30 ml). The pH was adjusted to 4.5 with glacial acetic acidand filtered. The filtrate was applied to a cation exchange (BioRex-70)column (H+ form), washed with water and then eluted with 100 ml ofpyridine acetate buffer (30 ml of pyridine, 4 ml of acetic acid, 66 mlof water). The eluant was evaporated to dryness. The residue wasdissolved in a small amount of 10% acetic acid and diluted with water to1% acetic acid, then lyophilized, yielding 650 mg of the crude titledpeptide.

The crude peptide was purified by counter current distribution inn-butanol/acetic acid/water (B/A/W) (4:1:5) yielding 33 mg partiallypurified peptide. This was further purified by gel filtration on aSephadex G-15 column in 1% acetic acid, yielding 24.5 mg pure peptide.Amino acid analysis (hydrolysis in HCl/TFA 2:1, 0.005% phenol for 1 hr.)Asp 1.00, Pro 0.72, Cys 0.62, Val 0.99, Tyr 1.04, Phe 1.04, Arg 0.95,71% peptide. HPLC: (40% acetonitrile/60% water/0.1% TFA), one peak,k'=5.2; (45% acetonitrile/55% water/0.1% TFA) k'=3.6; (gradient 20%acetonitrile, 5'; 20-50% acetonitrile, 20'; 50% acetonitrile, 5')k'=8.7, 97% pure. Tlc: rf 0.32 (B/A/W 1:1:1); 0.12 (B/A/W 4:1:1); 0.50(n-butanol/pyridine/acetic acid/water), 15:10:3:12).

The extracted peptidyl resin still contained peptide by amino acidanalysis, so it was extracted with 3×50 ml of DMF. The DMF wasevaporated to dryness and the residue dissolved in 10% HOAc, diluted to1% acetic acid and lyophilized, yielding an additional 260 mg ofpeptide. FAB mass spectrometry of this material gave a m/z 1079 whichcorresponds to M+H for the desired cyclic peptide.

EXAMPLE 13

Substituting a stoichiometric quantity of Boc-D-Phe for Boc-D-Tyr(Br-Z)at the 2 unit of the peptide synthesis of Example 1 gives ##STR25##

Substituting Boc-D-Val at the same position using thesplitting-oxidation reactions of Example 2 gives ##STR26##

Substituting Boc-D-Leu in Example 1 gives ##STR27##

Substituting β-mercapto-β,β-cyclotetramethylenepropionic acid (Tmp) forPmp in Example 5 gives ##STR28##β-Mercapto-β,β-cyclohexamethylenepropionic acid gives the Hmp¹derivative.

Substituting in Example 1 Boc-D-Nle at the 2 unit and D-Arg(Tos) at the8 unit gives ##STR29##

Substituting in Example 2 Boc-D-Cha at the 2 unit gives ##STR30##

Substituting in Example 1 Boc-α-aminophenylbutyric acid (Pba) at the 2unit gives ##STR31##

Substituting Boc-Lys(ClZ) in Example 3 for the protected Arg gives##STR32##

Other representative compounds which are prepared in like manner are:##STR33##

EXAMPLE 14 Parenteral Dosage Unit Compositions

A preparation which contains 0.5 mg of the cyclic octapeptide ofExamples 1 or 3 as a sterile dry powder for parenteral injection isprepared as follows: 0.5 mg of peptide amide is dissolved in 1 ml of anaqueous solution of 20 mg of mannitol. The solution is filtered understerile conditions into a 2 ml ampoule and lyophylized. The powder isreconstituted before either intramuscular or intravenous injection to asubject suffering from edema susceptible to anti-ADH mechanism ofaction. The injection is repeated as necessary, from 1-5 times daily orin continuous i.v. drug injection. Other octapeptides of this inventionare made up and used in like manner.

Nasal Dosage unit Compositions

30 Mg of finely ground octapeptide of this invention such as the productof Example 2 is suspended in a mixture of 75 mg of benzyl alcohol and1.395 g of a suspending agent such as a commercial mixture ofsemisynthetic glycerides of higher fatty acids. The suspension is placedin an aerosol 10 ml container which is closed with a metering valve andcharged with aerosol propellants. The contents comprise 100 unit doseswhich are administered intranasally to an edematous subject from 1-6times a day.

What is claimed is:
 1. A polypeptide compound of the formula: ##STR34##in which: X is D-Phe, D-Val, D-Leu, D-Ile, D-Nva, D-Pba, D-Nle, D-Cha,D-Abu, D-Met, D-Chg, D-Tyr, L-Tyr, D-aIle, D-Tyr(alk) or L-Tyr(alk);P isPhe or Phe(4'Alk); Y is NH₂, NHAlk, NHBzl or OH; A is Val, Ile, Abu,Ala, Gly, Lys, Cha, Nle, Phe, Leu, Chg or Nva; W is L-Pro, ΔPro, D-Proor, when Y is OH, a single bond; Z is D-Arg, L-Arg, D-Lys, L-Lys or,when Y is OH, a single bond; Q¹ and Q² are both a displaceable group;and Cap is a β,β-cycloalkylene propionic acid with S-Q¹ attached at theβ-position and having 5-7 units in the cycloalkylene chain, or esterderivatives thereof.
 2. The compound of claim 1 in which Q¹ and Q² areacetamidomethyl.
 3. The compound of claim 1 in which Q¹ and Q² areacetamidomethyl, Y is OH and Z is a single bond.
 4. The compound ofclaim 1 being S-ACM-Pmp-D-Tyr(Et)-Phe-Val-Asn-S-ACM-Cys-Pro-OBzl.
 5. Thecompound of claim 1 beingS-Acm-Pmp-D-Tyr(Et)-Phe-Val-Asn-S-ACM-Cys-Pro(OH).